Like all blood cells, T lymphocytes are constantly lost during life, and must be continuously replaced. The thymus is the primary site of de novo T lymphopoiesis. Microenvironmental conditions unique to the thymus induce a complex series of developmental events in multipotent, marrow-derived progenitors, including positive and negative control of proliferation, T lineage specification, functional T lineage asymmetry, self-restriction, self-tolerance, and cell death/survival signals. Significant progress has been made in understanding the signals the thymus provides to lymphoid progenitors to induce and control these events. However, the proliferation, differentiation, and/or survival of the non-lymphoid (stromal) components of the thymus are equally dependent on lymphoid cells, and the absence of lymphoid cells (e.g., congenital immunodeficiency diseases) results in athymia, even though the nascent stromal cells are functionally competent. The signals that lymphoid cells provide to induce stromal growth, differentiation, and/or survival are completely unknown. In this proposal, we propose to address this process in a comprehensive fashion. Using our recently devised method for global characterization of stromal gene expression in situ, we will analyze the dynamic response of stromal cells to the presence of lymphoid progenitors in an inducible model of thymic growth. We will then identify stromally-expressed genes that encode receptors, and thus may respond to the presence of lymphoid cells, with particular emphasis on those that change during specific phases of the growth response (induction, log phase, termination, steady-state). We will verify the presence of the cognate ligands for these receptors in lymphoid cells, using informatic methods as well as manual means of curation. Cognate receptor:ligand pairs will then be prioritized using a variety of criteria, including dynamic patterns of expression, known functional relevance (growth, differentiation, survival) in other tissues, the availability of existing genetic models, etc. Finally, the biological relevance of a few high-priority candidates will be tested, using appropriate stromal- receptor or lymphoid-ligand mutant mouse models. This project is expected to provide novel insights into an unexplored area of biology that will not only fuel a better understanding of this process, but will facilitate the development of more mechanistic studies to characterize the unique, two-way interactions that occur between lymphoid progenitors and stromal cells in the thymus. PUBLIC HEALTH RELEVANCE: The thymus is the primary organ where T lymphocytes are made, and thus is critical for normal immune function. Specialized cells inside the thymus (stromal cells) are responsible for instructing stem-like cells recruited from the bone marrow to undergo development into T lymphocytes. However, early in development, these stromal cells themselves require signals from immature lymphocytes to induce their own growth and development. In the absence of lymphocytes, for instance, as occurs in many pediatric immunodeficiency disorders, the thymus does not form. The goals of this project are to perform an in depth analysis of this very poorly understood two-way interplay between lymphocytes and stromal cells in the thymus, and how these processes shape the development of the immune system.